Statistical characterization and development of summary measures of non-normal distributions of nuclear morphometry data from prostate cancer cells for use as prognostic indicators

Nuclear morphometry (NM) uses image analysis to measure features of the cell nucleus which are classified as: bulk properties, shape or form, and DNA distribution. Studies have used these measurements as diagnostic and prognostic indicators of disease with inconclusive results. The distributional properties of these variables have not been systematically investigated although much of the medical data exhibit nonnormal distributions. Measurements are done on several hundred cells per patient so summary measurements reflecting the underlying distribution are needed.^ Distributional characteristics of 34 NM variables from prostate cancer cells were investigated using graphical and analytical techniques. Cells per sample ranged from 52 to 458. A small sample of patients with benign prostatic hyperplasia (BPH), representing non-cancer cells, was used for general comparison with the cancer cells.^ Data transformations such as log, square root and 1/x did not yield normality as measured by the Shapiro-Wilks test for normality. A modulus transformation, used for distributions having abnormal kurtosis values, also did not produce normality.^ Kernel density histograms of the 34 variables exhibited non-normality and 18 variables also exhibited bimodality. A bimodality coefficient was calculated and 3 variables: DNA concentration, shape and elongation, showed the strongest evidence of bimodality and were studied further.^ Two analytical approaches were used to obtain a summary measure for each variable for each patient: cluster analysis to determine significant clusters and a mixture model analysis using a two component model having a Gaussian distribution with equal variances. The mixture component parameters were used to bootstrap the log likelihood ratio to determine the significant number of components, 1 or 2. These summary measures were used as predictors of disease severity in several proportional odds logistic regression models. The disease severity scale had 5 levels and was constructed of 3 components: extracapsulary penetration (ECP), lymph node involvement (LN+) and seminal vesicle involvement (SV+) which represent surrogate measures of prognosis. The summary measures were not strong predictors of disease severity. There was some indication from the mixture model results that there were changes in mean levels and proportions of the components in the lower severity levels. ^

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Caracterización molecular del mutante riso 1508 de cebada : modificaciones transcripcionales y posttranscripcionales en el desarrollo de la semilla

La semilla es el principal órgano reproductivo de las plantas espermatofitas, permitiendo la dispersión de las poblaciones y asegurando su supervivencia gracias a su tolerancia a la desecación y a su capacidad para germinar bajo condiciones ambientales óptimas. El rendimiento y valor económico de los cereales, que constituyen la primera cosecha mundial, depende, en buena medida, de la eficacia con que se acumulan en la semilla sustancias de reserva: proteínas, carbohidratos y lípidos. El principal carbohidrato acumulado en la semilla de cebada es el almidón y la fracción mayoritaria de proteínas es la de las prolaminas (solubles en etanol al 70%); estas proteínas tienen muy bajo contenido en lisina, un aminoácido esencial en la dieta de animales monogástricos. Con el fin de mejorar el valor nutricional de la semilla de cebada, se han obtenido diferentes mutantes con un mayor contenido en este aminoácido. Riso 1508 es un mutante de cebada rico en lisina cuya mutación lys3a, de efectos pleiotrópicos, segrega como un único gen mendeliano. Entre otros, presenta una reducción drástica de la expresión de algunos genes que codifican proteínas de reserva de tipo prolamina, en concreto, presenta reducida la expresión de los genes que codifican B-, C- y ϒ-Hordeínas y del inhibidor de tripsina CMe, pero no tiene alterada la expresión del gen que codifica las D-Hordeínas. Este último gen carece en su promotor del motivo GLM (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), que es reconocido por factores transcripcionales bZIP. En este trabajo, el mutante de cebada Riso 1508 se ha utilizado como herramienta para profundizar en el conocimiento de la regulación génica en semillas durante las fases de la maduración y la germinación. Para ello, en una primera aproximación, se llevó a cabo un análisis transcriptómico comparando el genotipo mutante con el silvestre durante la maduración de la semilla. Además de confirmar variaciones en los genes que codifican proteínas de reserva, este análisis indicó que también estaban afectados los genes relacionados con metabolismo de carbohidratos. Por ello se decidió caracterizar la familia multigénica de sacarosas sintasa (SUSy) en cebada. Se anotaron dos nuevos genes, HvSs3 y HvSs4, cuya expresión se comparó con la de los genes HvSs1 y HvSs2, previamente descritos en el laboratorio. La expresión de los cuatro genes en tejidos diferentes y su respuesta a estreses abióticos se analizó mediante RT-qPCR. HvSs1 y HvSs2 se expresaron preferencialmente durante el desarrollo del endospermo, y HvSs1 también fue un tránscrito abundante durante la germinación. HvSs1 se indujo en hojas en condiciones de anoxia y HvSs3 por estrés hídrico, y ambos genes se indujeron por tratamientos de frío. La localización subcelular de las cuatro isoformas no fue sólo citoplásmica, sino que también se localizaron en zonas próximas a retículo endoplásmico y en la cara interna de la membrana plasmática; además, se observó una co-localización de HvSS1 con el marcador de mitocondrias. Estos datos sugieren un papel distinto aunque parcialmente solapante de las cuatro Sacarosa Sintasas de cebada, descritas hasta la fecha. Las cinéticas de expresión de los genes que codifican los TFs más importantes implicados en la regulación génica durante el desarrollo del endospermo de cebada, se analizaron por RT-qPCR en ambos genotipos, demostrando que los TFs de la clase DOF aparecieron desregulados durante todo el proceso en Riso 1508 comparado con el cv. Bomi, aunque también se observaron diferencias significativas en algunos de los que codifican bZIPs. Estudios previos indicaban que el ortólogo de BLZ2 en maíz, O2, se regula post-traduccionalmente mediante un mecanismo de fosforilación/defosforilación reversible, y que la forma defosforilada es la fisiológicamente activa. En este trabajo se demostró que BLZ2 está sujeto a este tipo de regulación y que la proteín-fosfatasa HvPP2C2 está implicada en el proceso. La interacción de HvPP2C2 y BLZ2 tiene lugar en el núcleo celular únicamente en presencia de 100 μM ABA. En el mutante Riso 1508, BLZ2 se encuentra en un estado hiperfosforilado tanto durante la maduración como durante la germinación de la semilla, lo que dificultaría la unión de BLZ2 a las secuencias GLM en los promotores de los genes que codifican B-, C-,y ϒ- Hordeínas y CMe. Summary The seed is the main reproductive organ of spermatophyte plants allowing the spread of populations and ensuring their survival through its desiccation tolerance and because of their ability to germinate under optimum environmental conditions. Yield and economic value of cereal crops, that constitute the first world crop, depend largely on the efficiency with which they accumulate in the seed reserve substances: proteins, carbohydrates and lipids. The main carbohydrate accumulated in the barley seed is starch and the major protein fraction is that of prolamins (soluble in 70% ethanol); these proteins have a very low lysine content, an essential amino-acid for the diet of monogastric animals. In order to improve the nutritional value of the barley seed, different mutants have been obtained with a higher content of this amino-acid. Riso 1508 is one lysine-rich mutant whose mutation (lys3a) segregates as a single Mendelian gene with pleiotropic effects, such as a drastic reduction of genes encoding the trypsin inhibitor CMe and the B-, C-and ϒ-hordeins, but has not altered the expression of the gene encoding the D-hordeins. This latter gene lacks in its promotor the GLM motif (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), that is recognised by bZIP transcription factors In this work we have used the barley mutant Riso 1508 as a tool for better understanding gene regulation in seeds during the maturation and germination phases. To this aim, a transcriptomic analysis was performed comparing wild and mutant genotypes during seed maturation. Besides confirming variations in the expression of genes encoding reserve proteins, this analysis indicated that some genes related with carbohydrate metabolism were also affected. It was therefore decided to characterize the multigene family of sucrose synthases (SUSy) in barley. Two new genes were annotated, HvSs3 and HvSs4, and its expression was compared with that of genes HvSs1 and HvSs2, previously described in our laboratory. The expression of the four genes in different tissues and in response to abiotic stresses was analyzed by RTqPCR. HvSs1 and HvSs2 were preferentially expressed during the development of the endosperm, and the HvSs1 transcript was also abundant upon germination. HvSs1 was induced in leaves by anoxic conditions, HvSs3 by water stress, and both genes were induced by cold treatments. The subcellular localization of all four isoforms was not only cytoplasmic, but they could be found along the endoplasmic reticulum and at the inner side of the cell membrane; HvSS1, was also associated with the mitochondrial marker. These data suggest a distinct but partially overlapping roles for the barley sucrose synthases, described so far. The expression kinetics of the genes encoding the most important TFs involved in gene regulation during barley endosperm development was analyzed by RT-qPCR in both genotypes. These data show that the genes encoding DOF TFs were mis-regulated throughout the process in Riso 1508, although significant differences were also found among some of those encoding bZIPs. Previous studies indicated that the BLZ2 orthologue in maize, O2, was post-translationally regulated by reversible phosphorylation/dephosphorylation and that the dephosphorylated protein is the physiologically active form. In this work we demostrate that BLZ2 is under a similar regulation and that the proteinphosphatase HvPP2C2 is implicated in the process. The interaction between HvPP2C2 and BLZ2 takes place in the cell nucleus only in the presence of 100 μM ABA. In the Riso 1508 mutant, BLZ2 is found in a hyperphosphorylated state in the maturation phase and upon seed germination; because of this, the BLZ2 binding to the GLM promoter sequences of genes encoding B-, C- y ϒ- Hordeins and CMe would be decreased in the mutant.

Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1− yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1− yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds

The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to β-glucuronidase (GUS) to study their activity pattern. The FIS2∷GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus. After pollination, the maternally derived FIS2∷GUS protein occurs in the nuclei of the cenocytic endosperm. Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst. MEA∷GUS has a pattern of activity similar to that of FIS2∷GUS, but FIE∷GUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination. The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development. Maternal and not paternal FIS2∷GUS, MEA∷GUS, and FIE∷GUS show activity in early endosperm, so these genes may be imprinted. When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage. The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent. The wild-type alleles of MEA or FIS2 are not required. The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

Expression of mouse telomerase catalytic subunit in embryos and adult tissues

Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing the stable maintenance of chromosomes during multiple cell divisions. Here, we describe the isolation and characterization of the catalytic subunit of mouse telomerase, mTERT (mouse telomerase reverse transcriptase), an essential protein component of the telomerase complex. During embryonic development, mTERT mRNA is abundantly expressed in the whole embryo, especially in regions of intense proliferation. We found that the mTERT mRNA expression in both embryonic and adult tissues is independent of the essential RNA component of telomerase, mTR, and therefore, of the formation of active telomerase complexes. mTERT protein is present exclusively in tissues with telomerase activity, such as testis, spleen, and thymus. mTERT protein is barely detectable in the thymus of mTR−/− mice, suggesting that mTERT protein stability in this tissue may depend on the actual assembly of active telomerase complexes. Finally, we found that mouse and human telomerase catalytic subunit is located in the cell nucleus, and its localization is not regulated during cell cycle progression.

HNS, a nuclear-cytoplasmic shuttling sequence in HuR

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3′ untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.

Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.

Ash1p is a site-specific DNA-binding protein that actively represses transcription

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.